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1 Laboratory of Microbiology, Department of Biochemistry, Physiology and Microbiology, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
2 BCCM/LMG Bacteria Collection, Department of Biochemistry, Physiology and Microbiology, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
3 Research Group of Industrial Microbiology and Food Biotechnology, Department of Applied Biological Sciences and Engineering, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
Correspondence
Ilse Scheirlinck
Ilse.Scheirlinck{at}ugent.be
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains LMG 23699T, LMG 23701, LMG 23702 and R-30103 are AM285450, AM285454, AM285451 and AM285452, respectively, and for the pheS gene sequences AM285025, AM285027, AM285028 and AM285449, respectively.
A figure showing data from numerical analysis of the protein profiles is available with the online version of this paper.
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). Both groups of micro-organisms, present in the flour or added as a starter, contribute to the sourdough fermentation. While yeasts are primarily responsible for the leavening process, LAB mainly contribute to lactic fermentation, proteolysis, synthesis of aromatic compounds, and anti-mould and anti-ropiness activity (Gobbetti, 1998; Hammes & Gänzle, 1998). Lactobacillus species dominate in sourdough due to their competitiveness and adaptation to the specific environmental conditions (De Vuyst & Neysens, 2005). The majority of recently described species isolated from sourdoughs are members of the Lactobacillus plantarum group and Lactobacillus brevis group, such as Lactobacillus nantensis (Valcheva et al., 2006) and Lactobacillus namurensis (Scheirlinck et al., 2007), respectively. The phylogenetic relatedness of species within these groups is strongly based on 16S rRNA gene sequence analysis (Rossello-Mora & Amann, 2001); however, in other bacterial taxa, the high degree of conservation of the 16S rRNA gene complicates the differentiation of such closely related species (Cooper & Feil, 2004). Recent work has indicated that the protein-encoding gene phenylalanyl-tRNA synthase (pheS) is a promising tool for delineation and identification of Lactobacillus species (S. M. Naser, P. S. R. Dawyndt, B. Hoste, D. Gevers, K. Vandemeulebroecke, I. Cleenwerck, M. Vancanneyt & J. Swings, unpublished; Vancanneyt et al., 2006). In the period 2002–2004, we obtained a group of Lactobacillus isolates from traditional Belgian wheat sourdoughs that could not be assigned to any hitherto known species. In the present study, phenotypic and genotypic evidence is presented to support the description of this group as a novel Lactobacillus species for which the name Lactobacillus crustorum sp. nov. is proposed.
The wheat sourdough samples used in this study originate from biodiversity studies performed at two different Belgian artisan bakeries. Both wheat sourdoughs were incubated at 28–30 °C and used for the production of cookies, baguettes, hard rolls and bread. Sourdough samples were taken aseptically, stored at 4 °C and analysed within 24 h. Sourdough samples were suspended (1 : 10, w/v) and serially diluted in peptone-physiological solution (PPS) [0.1 % (w/v) bacteriological peptone (Oxoid) and 0.85 % (w/v) NaCl]. Sample dilutions were incubated at 30 and 37 °C under aerobic and anaerobic conditions on MRS5 agar containing 0.1 g cycloheximide l–1 (Meroth et al., 2003). Colonies were picked, checked for bacteriological purity and stored in Microbank tubes (Pro-Lab Diagnostics) at –80 °C. Isolates LMG 23699T, LMG 23701, LMG 23702 and R-30103 were cultured in an aerobic atmosphere at 30 °C. Isolates LMG 23701 and LMG 23702 originate from a wheat sourdough produced in the province of Namur, Belgium, that was sampled in 2002. Isolates LMG 23699T and R-30103 were recovered from a wheat sourdough sampled in 2004 and 2005, respectively. The latter sourdough was produced in the province of Oost-Vlaanderen, Belgium. Gram-staining, cell morphology, catalase activity and all further experiments were performed with cells cultivated for 24 h on MRS5 medium and incubation at 30 °C under aerobic conditions, unless otherwise indicated.
The original collection of sourdough isolates obtained during the biodiversity studies was initially screened by means of rep-PCR fingerprinting. For rep-PCR analysis, total DNA was extracted from single colonies grown on MRS5 agar by alkaline lysis. A small portion of one colony was resuspended in 20 µl lysis buffer (2.5 ml 10 % SDS, 5.0 ml 1 M NaOH, 92.5 ml Milli-Q water). The mixture was heated at 95 °C for 15 min, cooled immediately on ice and after a short centrifugation at high speed, 180 µl sterile Milli-Q water was added. Subsequently, the mixture was centrifuged at 13 000 g for 5 min and stored at –20 °C. If the alkaline lysis method was not able to produce good quality rep-PCR profiles, a phenol/chloroform method was used, as described by Gevers et al. (2001). The rep-PCR oligonucleotide primer used in this study was (GTG)5 (5'-GTGGTGGTGGTGGTG-3') (Versalovic et al., 1994). The conditions for amplification and gel electrophoresis were as reported by Versalovic et al. (1994). The (GTG)5-PCR profiles were visualized after staining with ethidium bromide under UV light, followed by digital image capturing using a CCD camera. The resulting fingerprints were analysed using the BioNumerics V4.0 software package (Applied Maths). Isolates were tentatively assigned to a given species when high similarities between the (GTG)5-PCR fingerprints of isolates and reference strains of typical sourdough species were obtained. A separate (GTG)5-PCR cluster containing 11 sourdough isolates from two different bakeries could not be allocated to any species in the (GTG)5-PCR reference framework (data not shown). Four isolates from this cluster that produced a unique (GTG)5-PCR band pattern, i.e. LMG 23699T, LMG 23701, LMG 23702 and R-30103, were selected for further polyphasic characterization.
The taxonomic position of the four selected sourdough isolates was first investigated using pheS gene sequence analysis (Naser et al., 2005). Genomic DNA was extracted as described by Gevers et al. (2001). The primers for pheS sequencing were PheS-21-F and PheS-23-R, using amplification conditions and sequencing reactions as described by Naser et al. (2005). Sequences were imported into the BioNumerics V4.0 software, aligned and compared using the neighbour-joining method with available sequences of nearly all recognized Lactobacillus species. All four isolates constituted a separate sub-branch in the Lactobacillus plantarum group, showing sequence similarities below 90 % with the other members of the group (Fig. 1). Isolates LMG 23699T, LMG 23701 and R-30103 shared 100 % sequence similarity, whereas isolate LMG 23702 showed 90.2 % pheS sequence similarity with the other three sourdough isolates. Interspecies gaps in lactobacilli normally exceed 7 % (S. M. Naser, P. S. R. Dawyndt, B. Hoste, D. Gevers, K. Vandemeulebroecke, I. Cleenwerck, M. Vancanneyt & J. Swings, unpublished), which suggested that the four sourdough isolates represented one or possibly two new Lactobacillus species.
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). The obtained sequences (continuous stretches of 1519 bp) were deposited in, and aligned and clustered with sequences from the EMBL database. A phylogenetic tree was constructed using the neighbour-joining method and the BioNumerics V4.0 software package. Unknown bases were discarded for analysis. The statistical reliability of the tree was evaluated by bootstrap analysis of 500 replicates and the tree topology was confirmed using maximum-parsimony and maximum-likelihood. 16S rRNA gene sequence analysis indicated that the four sourdough isolates clearly belong to the Lactobacillus plantarum group. Isolate LMG 23699T is most closely positioned to Lactobacillus nantensis (LMG 23510T), Lactobacillus mindensis (LMG 21932T) and Lactobacillus farciminis (LMG 9200T), showing 16S rRNA gene sequence similarities of 98.4, 98.9 and 99.3 %, respectively (Fig. 2). All four new isolates shared more than 99.5 % 16S rRNA gene sequence similarity, which contrasted with the separate position of isolate LMG 23702 revealed by pheS sequence analysis.
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for Gram-positive bacteria. Densitometric digitization of patterns was performed using an LKB 2202 Ultrascan Laser Densitometer. Normalization of densitometric traces was performed using the GELCOMPAR version 4.2 software (Applied Maths). Numerical analysis with profiles available in an extensive in-house database was performed using the Pearson product moment correlation coefficient and the unweighted pair-group method using arithmetic averages (UPGMA) with BioNumerics V4.0 software. Among the protein profiles of the four sourdough isolates, notable differences were observed in the position and intensity of the dominant bands situated in a molecular mass range of 25–35 kDa. Numerical analysis of the protein profiles, omitting the latter zone, resulted in the delineation of one distinct branch for the four sourdough isolates (see supplementary Fig. S1 with the online version of this paper). Between isolates LMG 23699T and R-30103, only minor differences were observed in the intensity of dominant bands which could suggest that both isolates are subcultures of a diverging clone.
Further genotypic characterization and differentiation of the four new isolates was performed using amplified fragment length polymorphism (AFLP) fingerprinting of whole genomes. Extraction and purification of total genomic DNA was done as described above. AFLP analysis was performed according to the protocol of Thompson et al. (2001) with several modifications. Total DNA was digested with EcoRI and TaqI restriction enzymes. Fragments were amplified using the primers E01 (5'-GACTGCGTACCAATTCA-3') and T01 (5'-CGATGAGTCCTGACCGAA-3'). The resulting electrophoretic profiles were normalized using the GeneScan 3.1 software (Applera), and tables of peaks, containing fragments of 50–536 bp, were transferred into the BioNumerics V4.0 software. AFLP patterns were compared with profiles of LAB reference taxa using the Dice coefficient and UPGMA linkage. Cluster analysis of the AFLP band patterns confirmed the unique taxonomic position of isolates LMG 23699T, LMG 23701, LMG 23702 and R-30103. Also, we observed that the AFLP pattern of isolate LMG 23702 was the most divergent compared to the other three sourdough isolates. Although isolates LMG 23699T and R-30103 displayed highly similar fingerprints, AFLP analysis allowed strain differentiation among the four new isolates and thus excluded clonal relatedness (Fig. 3).
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. Genomic DNA was extracted as described above. The nucleoside mixture was separated by HPLC using a Waters SymmetryShield C8 column maintained at 37 °C. The solvent was 0.02 M (NH4)H2PO4 (pH 4.0) with 1.5 % acetonitrile. Non-methylated 
-phage DNA (Sigma) was used as the calibration reference. The DNA G+C contents of the strains LMG 23699T, LMG 23701, LMG 23702 and R-30103 were 35, 36, 35 and 35 mol%, respectively. These values are within the range reported for the Lactobacillus plantarum group (34–46 mol%) (Ehrmann et al., 2003).
DNA–DNA hybridizations were performed between strains LMG 23699T, LMG 23701, LMG 23702 and R-30103 and the type strains of the seven most closely related species, i.e. Lactobacillus farciminis, Lactobacillus nantensis, Lactobacillus mindensis, Lactobacillus kimchii, Lactobacillus paralimentarius, Lactobacillus alimentarius and Lactobacillus versmoldensis (Figs 1 and 2
). DNA was extracted from 0.75–1.25 g (wet wt) cells using the protocol described by Gevers et al. (2001) with the following modifications. Volumes were increased tenfold for large-scale application. After the addition of 20 % SDS and glass beads, cells were mixed for 30 s. Subsequently, 16.5 ml TE buffer (10 mM Tris/HCl, 100 mM EDTA, pH 8.0) and 5 ml 5 M NaCl were added, followed by 10 min incubation at 65 °C. The chloroform/isoamylalcohol extraction, and ethanol and RNase treatment were performed as described by Marmur (1961). DNA–DNA hybridizations were performed with biotin-labelled probes in microplate wells (Ezaki et al., 1989), using an HTS7000 Bio Assay Reader (Perkin Elmer) for the fluorescence measurements. The hybridization temperature was 34 °C in the presence of 50 % formamide. Reciprocal experiments were performed for every pair of strains and standard deviation values ranged from 0.3 to 12. DNA–DNA binding values between strain LMG 23699T and the type strain of Lactobacillus alimentarius (LMG 9187T), Lactobacillus farciminis (LMG 9200T), Lactobacillus nantensis (LMG 23510T), Lactobacillus paralimentarius (LMG 19152T), Lactobacillus mindensis (LMG 21932T), Lactobacillus kimchii (LMG 19822T) and Lactobacillus versmoldensis (LMG 21929T) were 35, 33, 31, 31, 30, 26 and 14 %, respectively. These values are well below the threshold of 70 % suggested for species delineation (Stackebrandt & Goebel, 1994), indicating that LMG 23699T represents a novel species in the genus Lactobacillus. Furthermore, DNA–DNA hybridizations were performed between strain LMG 23699T and strains LMG 23701, LMG 23702 and R-30103. Hybridization values were above the 70 % threshold (range 72–96 %), indicating that all strains belong to the same species. DNA–DNA binding values were lowest between strain LMG 23702 and strains LMG 23699T, LMG 23701 and R-30103 (range 72–77 %), which is in line with the slightly aberrant position of the former strain in pheS sequencing and AFLP analysis (Figs 1 and 3). Despite this, polyphasic taxonomic evidence collectively indicates that strain LMG 23702 is sufficiently close to the other three sourdough strains to be considered a member of the same species.
Growth characteristics of strains LMG 23699T, LMG 23701, LMG 23702 and R-30103 were determined in MRS broth (pH 5.4) (de Man et al., 1960). Growth was tested at 15 and 45 °C and in the presence of 5, 6 and 7 % NaCl. Aerobic and anaerobic growth and production of gas from 2 % glucose and 2 % gluconate in MRS-broth (pH 5.4, without triammonium citrate) was investigated. Arginine hydrolysis was tested in a medium containing 0.5 % tryptone, 0.5 % yeast extract, 0.3 % L-arginine, 0.05 % glucose and 0.2 % K2HPO4 (pH 7.0), with methyl orange as indicator. The isomeric type of lactate was determined enzymically (R-Biopharm). The carbohydrate fermentation patterns of the strains were determined using the API 50 CHL system (bioMérieux), following the manufacturer's instructions with strains cultivated at 37 °C. A detailed phenotypic description is given below and characteristics differentiating the new species from its closest relatives Lactobacillus farciminis, Lactobacillus nantensis and Lactobacillus mindensis are summarized in Table 1.
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Description of Lactobacillus crustorum sp. nov.
Lactobacillus crustorum (crus.tor'um. L. gen. pl. n. crustorum, of breads/cakes).
Cells are Gram-positive, catalase-negative, non-motile and non-spore-forming. The rod-shaped cells occur singly or in pairs, are 2–15 µm in length and 0.5–1 µm wide. After 24 h incubation on MRS5 agar, colonies are beige, slightly irregular, convex with a slightly rough surface and approximately 0.5–2 mm in diameter. The cells grow well in liquid or solid MRS under aerobic or anaerobic conditions. The strains grow at 15 °C in the presence of 5, 6 and 7 % NaCl and at 45 °C. All strains produce more than 90 % of the L-lactate isomer and glucose is metabolized homofermentatively. Ammonium is not produced from arginine. No gas is produced from glucose or gluconate. All strains produce acid from galactose, glucose, fructose, mannose, N-acetylglucosamine, aesculin and salicin, but not from glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, 
-methyl-D-xyloside, sorbose, dulcitol, inositol, mannitol, sorbitol, 
-methyl-D-mannoside, -methyl-D-glucoside, melibiose, sucrose, inulin, melezitose, raffinose, starch, glycogen, xylitol, D-turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Acid production from rhamnose, amygdalin, arbutin, cellobiose, maltose, lactose, trehalose, gentiobiose and D-tagatose is strain-dependent. The DNA G+C content is 35–36 mol%.
The type strain, LMG 23699T (=CCUG 53174T), was isolated from an artisan wheat sourdough produced in the province of Oost-Vlaanderen, Belgium.
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ACKNOWLEDGEMENTS |
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